This invention relates to new protecting groups for asparagine and glutamine in solid phase peptide synthesis and more particularly to trialkoxy benzyl protecting groups for protection of the asparagine and glutamine residues. Solid phase peptide synthesis typically begins with covalent attachment of the carboxyl end of a first alpha-amine protected acid through an organic linker to an insoluble resin synthesis bead. This can be illustrated as: ##STR2## wherein .circle.P is the insoluble synthesis resin, Aa.sub.1 is the first amino acid and X is a protecting group such as Fmoc, t-Boc and the like.
The general synthesis cycle then consists of deprotection of the alpha-amine group of the last amino acid, washing and, if necessary, neutralization, followed by reaction with a carboxyl activated form of the next alpha-amine protected amino acid to be added. The peptide chain then becomes: ##STR3## wherein Aa.sub.2 is the second amino acid. The cycle is repeated to the nth amino acid to yield: ##STR4## wherein Aa.sub.n is the n.sup.th amino acid.
Each successive amino acid is attached to the terminal nitrogen by the carbonyl carbon of the carboxylic acid group. Addition of asparagine and glutamine acid residues present particular problems because each have an amide side chain in addition to the amino acid group. The structural formulae are as follows: ##STR5## wherein asparagine is shown when n is 1 and glutamine when n is 2.
FIG. 1 is a diagrammatic representation of a peptide synthesis apparatus suitable for automated computer controlled solid phase synthesis. Such apparatus are available from Biosearch, Inc. of San Rafael, Calif.
Present automatic peptide synthesizers conventionally involves preactivation of a protected amino acid utilizing Diisopropylcarbodiimide (DIPCDI). The synthesis is carried out in a reaction vessel 11 which includes a synthesis resin 12 therein. Reaction vessel 11 is coupled to a source of protected amino acid 13 and a source of DIPCDI activator 14, in a solvent such as CH.sub.2 Cl.sub.2. Protected amino acid from amino acid reservoir 13 is fed to reaction vessel 11 through a line 18 by an amino acid control valve 16 and DIPCDI activator is fed into line 18 and mixed with the amino acid by an activator valve 17. Amino acid valve 16 and activator valve 17 are activated in brief alternate intervals so that protected amino acid and DIPCDI activator are mixed in line 18 for a preselected time prior to being fed into reaction vessel 11.
After the coupling reaction under a nitrogen atmosphere in reaction vessel 12 is complete, the protected amino acid now coupled through its carboxylic acid group to synthesis resin 12 is deblocked with, for example trifluoroamine (TFA), washed with a base and the next activated amino acid residue is added to reaction vessel 11. Upon obtaining the desired peptide residue, the peptide is cleaved from synthesis support 12, generally with hydrofluoric acid (HF).
In conventional t-Boc solid phase peptide synthesis, addition of asparagine and glutamine is performed using diisopropylcarbodiimide (DIPCDI) or dicyclohexylcarbodiimide (DCCI) coupling in the presence of 1-hydroxybenzotriazole (HOBt). The protocol is: ##STR6##
When the derivatives and additives are dissolved in dimethylformamide (DMF) at 0.4M concentration and mixed in-line with DIPCDI, no precipitation occurs and couplings proceed well without significant dehydration of the amide side chains to the corresponding nitriles. Xanthenyl derivatives, on the other hand, are less soluble and the active intermediates crystallize rapidly during in-line mixing causing poor coupling and clogging of valves in automated synthesizers.
Alternatively, Fmoc mediated solid phase peptide synthesis can be performed using the following protocol: ##STR7##
Unprotected derivatives of asparagine and glutamine are very insoluble in Fmoc mediated solid phase peptide synthesis. Only a 0.2M solution of Fmoc-Asn-OH in DMF can be prepared and precipitation occurs when the Fmoc protected asparagine is mixed with DIPCDI and HOBt. In the case of Fmoc protected glutamine, complete solution is not effected even after prolonged sonication at 0.2M.
The use of pentafluorophenyl esters to increase solubility has been proposed and is effective in the case of Fmoc protected asparagine resulting in good coupling. However, Fmoc-Gln-OPFP is still completely insoluble and poor coupling is observed. Furthermore, sampling of stored DMF solutions of active esters is not possible because racemisation, dehydration and dimer formation occur albeit at slow rates. This slow dissolution and poor solubility of the active esters complicates operation and limits performance.
Sequences containing Asn-X and Gln-X wherein X is a non-hindered amino acid residue tend to form cyclic imides under acidic or basic conditions. These cyclic imides can open to lead to deamidated alpha and beta peptides. In slow couplings containing N-terminal unprotected Gln, pyroglutamyl formation gives rise to a significant amount of chain termination. Sequences rich in Asn and Gln are formed at slow rates due to the tendency for interpeptide hydrogen bonding to occur causing interpeptide aggregation and reduced coupling efficiency. Such interpeptide hydrogen bonding sterically masks the amino groups.
Other problems include the occurrence of dehydration side reactions on activation that result in nitrile containing byproducts. Finally, the poor solubility of these derivatives even in DMF is the most serious problem and is just as apparent with pentafluorophenyl and other active ester derivatives as with the free acids themselves.
These problems are also directly applicable to Dts mediated syntheses even though some of the side reactions are minimized under neutral conditions. Dts-Asn-OH and Dts-Gln-OH are only slightly soluble in inorganic solvents resulting in yields from polyethylene glycol xanthate mediated syntheses of only about 20%.
Attempts have been made to protect the amide side chain using dimethoxybenzhydryl protecting groups (Mbh). However, Mbh protection provides only poor yields and requires relatively drastic cleavage conditions.
Accordingly, it is desirable to provide an improved protecting group for use on the amide side chain in Asn and Gln in solid phase peptide synthesis.